Thyroid hormone receptors (TRs) belong to a superfamily of nuclear hormone receptors including the steroid hormone, vitamin D, and retinoid receptors. They can bind as heterodimers with retinoid X receptors to hormone response elements (HREs) located in the promoter region of target genes. For positively-regulated genes, thyroid hormone (T3) binds to the receptor and increases transcription. Interestingly, in the absence of T3, TRs can still bind to HREs and repress transcription below basal level. There is emerging evidence on how the receptor mediates these effects. Recently, several groups have described co-activators and co-repressors that interact with the receptor in a ligand-dependent manner. Additionally, there are other proteins that interact with the liganded receptor/co-activator complex such as CBP and p/CAF which have intrinsic histone acetylase activity. Similarly, co-repressors have been shown to complex with histone deactylases. We have several ongoing projects to examine TR action at the molecular level: repressor interactions with TR and co-factor shuttling using this technology. 1) We have examined the effects of T3 and T4 on in vivo hepatic gene regulation in mice using microchip array analyses. We have identified approximately 60 genes regulated by thyroid hormone, most of which have not been described previously. Interestingly, over half of the genes are negatively-regulated. Thus far, there have not been any studies of in vivo gene regulation described for nuclear hormone receptors. A manuscript describing this work was published in Molecular Endocrinology. We also are using similar methods to examine TR knockout mice in which TR isoforms have been selectively eliminated. 2) We have created a transgenic mouse line in which a dominant negative form of the co-repressor, N-CoRi, has been overexpressed in liver. Thus far, there have been no in vivo models of co-repressor effects on basal transcription of target genes. We observed that a number of target genes have increased basal transcription in hypo-and euthyroid transgenic mice suggesting that the co-respressors can play an improtant modulatory role for these genes in the hypo-and euthyroid states. We also used cDNA microarray to identify genes regulated by NCoR which are involved in cell proliferation. This work has been submitted. Dr. Feng made oral presentations of this work at the Endocrine Society Meeting and International Congress of Endocrinology. 3) We are using green fluorescent fusion proteins of mutant thyroid hormone receptors to study the effect of ligand-binding, co-repressor binding, DNA-binding, and heterodimerization on nuclear/cytoplasmic distribution in living cells by confocal microscopy. We have found evidence for active shuttling of TRs between the nuclear/cytoplasmic compartments in the absence and presence of T3. Additionally, interaction with RXR and N-CoR are important for nuclear retention of unliganded TR. Ligand-binding promotes nuclear reorganization of TRs with the formation of a speckling pattern. We have submitted a manuscript describing these findings. Dr. Maruvada presented her findings in oral presentations at the American Thyroid Association meeting and Endocrine Society Meetings. 4) We have identified novel alternatively-spliced and point mutant TRs in TSHoma pituitary samples. A manuscript has been submitted. 5) We have used yeast two-hybrid system to identify novel co-factors that interact with specific TR isoforms, and currently are characterizing them.